A Highly Conserved Cholesterol Metabolic Pathway in Mycobacterium Smegmatis Mc2155 And

A highly conserved cholesterol metabolic pathway in Mycobacterium smegmatis mc2155 and Mycobacteriumtuberculosis H37Rv

Esther García-Fernández,1 Daniel J. Frank,2 Beatriz Galán, 1 Petrea M. Kells,3 Larissa M. Podust,3José L. García,1 and Paul R. Ortiz de Montellano2

1Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain; 2Department of Pharmaceutical Chemistry, University of California, San Francisco, CA USA; 3Department of Pathology and Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, CA USA

Address editorial correspondence to:

Paul R. Ortiz de Montellano

University of California, San Francisco

600 16th Street, N576D

San Francisco, CA 94158-2517

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Running title: Cholesterol metabolism in Mycobacterium smegmatis

Summary

Degradation of the cholesterol side-chain in M. tuberculosisis initiated by two cytochromes P450,CYP125A1 and CYP142A1, thatsequentially oxidize C26 to the alcohol, aldehyde and acid metabolites.Here we report characterization of the homologousenzymes CYP125A3 and CYP142A2 from M. smegmatis mc2 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4-cholesten-3-one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4-cholesten-3-one to the C-26 alcohol and subsequently to the acid. The X-ray structures ofboth substrate-free CYP125A3 and CYP142A2 and of cholest-4-en-3-one-bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion of cyp125A3 or cyp142A2 does not impair growth of M. smegmatis mc2 155 on cholesterol. However, deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatis∆cyp125∆cyp142 double mutant retains its ability to grow on cholesterolalbeit with a diminished capacity, indicating an additional level of redundancy within its genome.

Introduction

Cytochromes P450 (CYPs) form a widely distributed class of heme-containing monooxygenases that are present in all domains of life. Actinobacteria genomes possess an unusually high number of CYPs (e. g., 20 CYPs in Mycobacterium tuberculosis H37Rv, 29 CYPs in Rhodococcus jostii RHA1 and 40CYPs in Mycobacterium smegmatis), in contrast to the absence of CYPs in Escherichia coliandin comparison to the 57 CYPs in the 3.3 Gb human genome (Ouellet et al., 2010a; Hudson et al., 2012). The typical function of CYPs is to catalyze the oxidation of organic substrates via their heme prosthetic group. This mono-oxygenation reaction involves the insertion of one oxygen atom from molecular oxygen into the substrate, while the second oxygen undergoes reduction to water (Ouellet et al., 2010a). The key stages of the catalytic cycle are: (i) substrate entry into the distal active site, which displaces the weakly iron-co-ordinated water present in the CYP resting state, (ii) heme-iron reduction from the ferric to the ferrous state by an electron-transport partner system and binding of molecular oxygen to the ferrous iron, (iii) a further single electron reduction of the oxy-complex and two protonation steps to release one oxygen as water and concomitantly generate a highly reactive intermediate iron(IV)-oxo porphyrin π-radical cation ([FeIV=O]+•), termed Compound I, and finally (iv) incorporation of the oxygen atom from Compound I into the substrate via a radical rebound mechanism (Denisov et al. 2005; Johnston et al. 2011).

Cholesterol and related steroids are ubiquitous throughout the environment due to their presence in cytoplasmic membranes and their role as precursors of vitamin D, the bile acids and all the sterol hormones. The microbial degradation of cholesterol proceeds through two biochemical stages: sterol side-chain elimination and steroid ring opening (Van der Geize and Dijkhuizen, 2004). The first step in cholesterol ring opening is the transformation of cholesterol into cholest-4-en-3-one that can be catalyzed in M. smegmatis by at least two different enzymes encoded by the genes MSMEG_5228 and MSMEG_5233, respectively (Uhía et al., 2011). Both enzymes are 3β-hydroxysteroid dehydrogenases belonging to the short-chain dehydrogenase/reductase (SDR) superfamily that binds NAD(P)(H) with a Rossman fold motif (Oppermann et al., 2003). The protein encoded by the MSMEG_5228 gene is very similar to the cholesterol dehydrogenases from Nocardia sp. and M. tuberculosis (Rv1106c) (Horinouchi et al., 1991; Yang et al., 2007), while the protein encoded by the MSMEG_5233 gene is similar to the AcmA dehydrogenase from Sterolibacterium denitrificanswhich is an O2independent hydroxylase that belongs to the dimethyl sulfoxide dehydrogenase molybdoenzyme family (Chiang et al., 2008; Dermer and Fuchs, 2012).

Recent data demonstrate that two key enzymes, CYP125 and CYP142 initiate cholesterol side-chain degradation in M. tuberculosis and R. jostii RHA1 (McLean et al., 2009; Capyk et al., 2009; Rosloniec et al., 2009; Ouellet et al., 2010b). These P450s perform sequential oxidations of the cholesterol side-chain at the C26 position, forming first the terminal alcohol, then the aldehyde, and finally the acid. This activity ultimately enables β-oxidation of the cholesterol side-chain (Ouelletet al.,2011; McLean et al., 2012). CYP125, the major P450 involved in side-chain oxidation in M. tuberculosis, is located in the igr operon, which is also important for M. tuberculosis survival in macrophages (Chang et al., 2009).An essential role of CYP125 for growth on cholesterol and alleviation of the toxicity of the cholest-4-en-3-one intermediate was observed with the CDC1551 strain of M. tuberculosisand in Mycobacterium bovis (BCG), but was not seen with the H37Rv strain, suggesting that the latter possesses one or more compensatory enzymes that allow it to cope in the absence of CYP125 (Ouellet et al., 2010b; Capyk et al., 2009). CYP142 can compensate for a deficiency of CYP125 and, in certain M. tuberculosis strains, cooperates with CYP125 in cholesterol catabolism (Driscoll et al., 2010; Johnston et al., 2010). Both cytochromes are able to oxidize the aliphatic side-chain of cholesterol or cholest-4-en-3-one at C-26to the carboxylic acid. CYP125 generates oxidized sterols of the (25S) configuration, whereas the opposite (25R) stereochemistry is obtained with CYP142 (Johnstonet al., 2010).

The CYP125 orthologue in M. smegmatis, a rapid-growing mycobacterium originally isolated from human smegma and commonlyfound in soil and water, is encoded by the MSMEG_5995 gene located within the MSMEG_5995_5990 putative operon. This operon has been suggested to be involved in cholesterol side-chain oxidation based on transcriptomic data and on its similarity with the igr operon from M. tuberculosis (Uhía et al., 2012). The CYP142 orthologue in M. smegmatis is encoded by the MSMEG_5918 gene located within the cholesterol gene cluster 2 that is induced in the presence of cholesterol (Uhía et al., 2012).The KstR regulator negatively controls the expression of both MSMEG_5995 and MSMEG_5918 genes (Kendall et al., 2007).

In this work, we report biochemical and structural characterization of the enzymes CYP125 and CYP142from M. smegmatis mc2 155 and analyze their role in the metabolism of cholesterol by constructing appropriatedeletion mutants. Whereas CYP142 serves as the sole back up for CYP125 in M. tuberculosis for the oxidation of the cholesterol side-chain, the cyp125cyp142 mutant of M. smegmatis retains its ability to utilize cholesterol as a carbon source, implying the presence of an additional level of redundancy within its genome.

Results

Cyp125 and Cyp142 genome region comparisons in M. tuberculosis and M. smegmatis

The cholesterol degradation pathway of cholesterol is highly conserved within the Actinobacteria, and particularly in the genus Mycobacterium.Bioinformatic analysis (TBLASTN) revealed that most of the mycobacterial genomes that are completely sequenced present a putative CYP125A3 and CYP142A2 orthologues (≥ 70% of identity), except for M. kansasii and M. leprae TN.

CYP125A3 (MSMEG_5995) from M. smegmatis shows a high amino acid sequence identity with CYP125A1 from M. tuberculosis (Rv3545c) (77%) (Table 1). MSMEG_5995 is located in the MSMEG_5990-MSMEG_5995 operon (Fig. S1) which shows a high identity (77-84%) with the igr operon (Rv3545c-Rv3540c) of M. tuberculosis that encodes an incomplete β-oxidation pathway for cholesterol metabolism (Table 1) (Mineret al., 2009; Thomas et al., 2011) and that is essential for survival of the pathogen (Sassetti and Rubin, 2003). The rest of the annotated functions within this operon are a lipid transfer protein (lpt2/MSMEG_5990/Rv3540c), two MaoC-like hydratases (MSMEG_5991/Rv3541c and MSMEG_5992/Rv3542c) and two acyl-CoA dehydrogenases (fadE29/MSMEG_5993/Rv3543c and fadE28/MSMEG_5994/Rv3544c). Directly opposite to MSMEG_5995 is located an acetyl-CoA acetyl transferase (fadA5/MSMEG_5996/Rv3546) that has been recently described as essential for utilization of cholesterol as a sole carbonsourceand for full virulence of M. tuberculosis in the chronic stage of mouse lung infection (Nesbitt et al, 2010).

MSMEG_5918 coding for CYP142A2 is located in the M. smegmatis cholesterol regulon outside the igr like region (Uhía et al., 2012) and shares high amino acid sequence identity with CYP142A1 from M. tuberculosis (Rv3518c) (78%) (Table 1). The annotated functions of the genes within this genomic region are an acyl-CoA synthetase (fadD19/MSMEG_5914/Rv3515c), an enoyl-CoA hydratase (echA19/MSMEG_5915/Rv3516c), three hypothetical proteins (MSMEG_5917/Rv3517c, MSMEG_5919/Rv3519 and MSMEG_5921/Rv3521), a coenzyme F420-dependent oxidoreductase (MSMEG_5920/Rv3520c), a lipid-transfer protein (lpt4/MSMEG_5922/Rv3522) and an acetyl-CoA acetyltransferase (lpt3/MSMEG_5923/Rv3523) (Table 1). The organization of the genomic region encoding CYP142in M. smegmatis is very similar to that of M. tuberculosis(Fig. S1). The only difference detected is the transcriptional direction of the MSMEG_5919 gene, which is opposite for the orthologue in M. tuberculosis. This change could modify the transcriptional regulation of the cyp142 gene since there is a KstR1 operator sequence in the MSMEG_5919-MSMEG_5920 intergenic region. The gene products around thecyp142 gene have been suggested to be involved in cholesterol side degradation in M. tuberculosis. Recent data demonstrated that FadD19 is a steroid CoA ligase essential for degradation of C-24 branched sterol side-chains in Rhodococcus rhodochrousDSM43269(Wilbrink et al., 2011).

Purification and spectral features of CYP125A3 and CYP142A2

Recombinant CYP125A3 and CYP142A2 from M. smegmatis were heterologously expressed in E. coli DH5α using the pCWOri+ vector. As stated in Experimental Procedures, they were purified to homogeneity by immobilized metal ion affinity chromatography followed by two steps of ion exchange chromatography yielding 35 and 43 mg purified protein per liter of harvested culture, respectively. SDS-PAGE analysis indicated that both CYP125A3 and CYP142A2 constituted >99% of the protein in the purified sample. CYP125A3 displayed spectral properties typical for a ferric P450 with most of the heme-iron in a high spin (HS) state with a Soret band at 393 nm and a small shoulder at 415 nm (corresponding to a low spin state) (Fig. 1A, solid line). This is consistent with the spectroscopic properties of CYP125 found by Ouellet et al., 2010b; Capyk et al, 2009 and McLean et al., 2009. In contrast, CYP142 has all its heme in the low spin state (LS), exhibiting a Soret band at 418 nm and the smaller α and β bands at 567 nm and 536 nm respectively, as it was described for CYP142 from M. tuberculosis (Driscoll et al., 2010) (Fig. 1B, solid line).

Reduction with sodium dithionite in the presence of carbon monoxide results in the formation of Fe2+-CO complexes giving CO-difference spectra with peaks at 451nm and 449 nm for CYP125A3 and CYP142A2, respectively (Fig. 1A and 1B, insets). Both proteins have a secondary peak at 422 nm, which is more evident in the case of CYP125,revealing the population ofthe P420 form of each isozyme.

CYP125A3 and CYP142A2 bind cholesterol, cholest-4-en-3-one and antifungal azole drugs

Binding of steroid ligands and antifungal azole drugs to CYP125A3 and CYP142A2 was studied by measuring the changes in the optical absorption spectra. Fig. 1 shows the absolute Soret region absorption spectra of CYP125 (Fig. 1A) and CYP142 (Fig. 1B) after incubation with cholesterol 50 µM (dotted line) and econazole 50 µM (dashed line). In contrast to the case of CYP125 and CYP142 from M. tuberculosis (Ouellet et al., 2010b; Johnstonet al., 2010), the addition of cholesterol to CYP125A3 and CYP142A2 results in complete conversion to the HS form as a result of the displacement of the water molecule coordinated to the heme iron atom. Due to the predominantly HS resting state of CYP125A3 only small changes are observed in the optical spectrum when cholesterol is added, notably an increase of the Soret band at 393 nm and a strong decrease of the shoulder at 415 nm (Fig. 1A). In the case of CYP142A2, the addition of cholesterol results in a clearType I shift of the Soret band from 418 nm to 393 nm (Fig. 1B). As observed previously for M. tuberculosis CYP125 and CYP142 (Driscollet al., 2010; Ouellet et al., 2010a), several antifungal azoles bind to CYP125 and CYP142, inducing a Type II spectral shift. Coordination of econazole to each protein causes a partial conversion of CYP125 to the LS state, resulting in a Type II shift of the Soret band to 415 nm (Fig. 1A), and a complete conversion of CYP142, inducing a Soret shift to 422 nm (Fig. 1B).

The KD values of CYP125A3 ad CYP142A2 for cholesterol, cholest-4-en-3-one and several antifungal azoles (miconazole, econazole and clotrimazole) were obtained from the spectral titration curves. The plots of the induced spectral change versus the steroid concentration (Fig. 2) and the azole concentration (Fig. S2) were fitted to a quadratic tight binding equation (Equation 1, see “Experimental procedures”) to generate the KD values that are listed in Table 2. For comparison, the literature KD values for CYP125 and CYP142 from M. tuberculosis are also included.

CYP125A3 and CYP142A2 catalyze the monooxygenation of C-26 steroids.

The enzymatic activities of the two M. smegmatis P450s in this study were examined in vitro using the heterologous electron donor partners, spinach ferredoxin and ferredoxin reductase, and an NADPH regenerating system. We observed the oxidation of cholest-3-en-4-one after 5 and 20 min of incubation with both CYP125A3 and CYP142A2, as judged from the appearance of new peaks in the HPLC chromatograms. The relative retention times (Rt) and mass spectra were consistent with production of 26-hydroxycholest-4-en-3-one (Rt 3.14 min, M+ 401)by both enzymes. The subsequent oxidation to cholest-4-en-3-one-26-oic acid (Rt 2.15 min, M+ 415) via the aldehyde cholest-4-en-3-one-26-al (Rt 4.53 min, M+ 399) was observed in assays with both enzymes, although it was more prevalent in assays using CYP142A2. The assignments of these products were based on their relative retention times and analysis of their mass spectra, which exhibit diagnostic peaks that match earlier assignments (Fig. S3) (Ouelletet al, 2010b; Johnstonet al. 2010).

Steady-state kinetics were measured and the parameters fit to the Michaelis-Menten equation (see Experimental Procedures), for the oxidation of cholent-4-en-3-one to 26-hydroxycholest-4-en-3-one. CYP125A3 and CYP142A2 showed similar KMvalues of 14.0 and 10.3 M, respectively; however, the overall rate of catalysis by CYP142A2 was approximately twice that of CYP125A3 (Fig. 3).

Overall structure of CYP125.

Consistent with the 77% sequence identity between two proteins, the 2.0 Å X-ray structure of M. smegmatisCYP125A3determined in this work is rather similar to that of the M. tuberculosis homolog, particularly when the substrate-free structures are compared (Fig. 4A). Although the substrate–bound form of CYP125A3 has not yet beencharacterized, the substrate position can be reasonably inferred from that of the M. tuberculosis CYP125A1-cholest-4-en-3-one complex (Ouellet et al, 2010b). Based on this approximation, residues contacting the cholest-4-en-3-one aliphatic side-chain are invariant. The conserved amino acid substitutions near the ring system include W83, M87 and L94 in M. smegmatis,compared with F100, I104 and V111 in M. tuberculosis, respectively. All three residues are bulkier in M. smegmatisand are situated on the flat side of the ring system. The most notable difference between the two binding sites is the lack of the D108-K214 salt-bridge interaction guarding the entrance to the active site at van der Waals distance to the substrate keto group in M. tuberculosis. Both residues are represented by alanine in M. smegmatis.

Overall structure of CYP142.

Although sharing a sequence of 78%, differences have also been observed between the CYP142 counterparts from M. tuberculosis and M. smegmatis. The substrate-freeM. smegmatis CYP142A2 is captured in a more “open” conformation than the M. tuberculosis enzyme due to repositioning of the F- and G-helices (Fig. 4B). Each protein chain in substrate-free CYP142A2 is associated with a molecule of -methyl cyclodextrin used to deliver the steroid substrate to the active site. Although the substrate failed to enter the active site in this crystal form, -methyl cyclodextrin molecules bound in the symmetry-related or special positions in the crystal, were unambiguously distinguished by donut-shaped electron densities. Due to lack of true lateral symmetry, all six are represented by alternative conformations, one flipped relative to the other. The untraceable bulk of electron density in the middle of each “donut” may belong to cholest-4-en-3-one cargo molecules.

The cholest-4-en-3-one-bound CYP142A2 crystals were obtained from an alternative set of crystallization conditions (Table 3). In the 1.69 Å CYP142A2 structure, the substrate was unambiguously defined by the electron density in a single binding orientation (Fig.5) in all four molecules in an asymmetric unit. Substrate-bound CYP142A2 was in a more open conformation than the substrate-free form, with the G-helix positioned further from the protein core to provide support for the substrate molecule. As in the CYP125 counterpart, the residues contacting the cholest-4-en-3-one aliphatic side-chain are invariant. Three amino acid substitutions, all of bulkier residues, including L72M75, M74Y77 and M222F255, distinguish the substrate binding sites of M. smegmatis CYP142A2 vs M. tuberculosis CYP142A1. Again, all three are clustered along the flat face of the sterol tetracyclic fusedring system(Fig. 5A). To accommodate bulkier side-chains, cholest-4-en-3-one is bent away from the triad (Fig. 5B). Distances between the heme iron and the carbon atoms of the branched methyl groups are 4.1Å and 5.6 Å, favoring formation of a product with R-configuration at C25.

Cholest-4-en-3-onebinding in CYP125 and CYP142.

The most striking observation comes from a comparison of the CYP125A1 and CYP142A2 substrate-bound forms. Mapping the amino acid contacts within 5 Å of the substrate on sequence alignments demonstrated that topologically identical residues constitute substrate binding sites in both proteins (Fig. 6). Yet, a 10-amino acid insert (102-111) after -strand3 adopts a helical structure in the crystal and provides extensive contacts that completely shield the substrate from bulk solvent in CYP125A1 (Fig. 7 A). The helical structure of this short fragment is stabilized by yet another insert in the CYP125 sequence (57-67), which is missing from CYP142 (Fig.6). Loss of both inserts in CYP142 results in exposure of the carbonyl group of cholest-4-en-3-one to the surface (Fig. 7B). This remarkable difference in the active site topology suggests that in contrast to CYP125, CYP142 may operate on C3-modified steroids substrates, such as cholesteryl esters of fatty acids or cholesterol sulfate.

CYP125 and CYP142 are not essential for the growth of M. smegmatis on cholesterol and cholest-4-en-3-one.

The roles of CYP125A3 and CYP142A2 in cholesterol catabolism were also investigated by mutagenesis of their respective coding genes, i.e., MSMEG_5995 and MSMEG_5918. The duplication rates (td) of the single mutant strains Cyp125(20 h) and Cyp142(21 h) were very similar to that the wild-type strain (21 h) when grown on cholesterol. However, the td of strain Cyp125 on cholest-4-on-3-one (17 h) increased in 4 h with respect to the wild-type and Cyp142 (13 h), presenting an initial lag phase of approximately 48 h. Growing on cholesterol or cholest-4-on-3-one was highly impaired for the double mutant strain Cyp125Cyp142, with higher duplication rates (25 h on cholesterol and 39 h on cholest-4-on-3-one) and longer lag phases (Fig. 8A and B). When the double mutant strain was complemented with cyp125A3 (pMVCyp125), this strain grew normally on cholesterol and cholest-4-on-3-one, giving duplication rates similar to the wild-type strain (16h on cholesterol and 13 h on cholest-4-on-3-one) (Fig. 8C). The production of the CYP125A3 protein in the complemented double mutant strain is shown in Fig. 8D. These results suggest that, although CYP125A3is the main enzyme responsible for the transformation of cholesterol and cholest-4-on-3-one into their oxidized metabolites, CYP142A2 supports the growth in absence of CYP125A3. The capacity of the double mutant to grow on both steroids suggests that at least one other cytochrome P450 encoded in the M. smegmatis genome is able to perform this biochemical step, or that an alternative cholesterol degradation pathway can be induced.